Drug Delivery
A variety of methods and routes of administration have been developed to deliver pharmaceuticals that include small molecular drugs and biologically-active compounds such as peptides, hormones, proteins, and enzymes to their site of action. Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal and intralymphatic injections that use a syringe and a needle or catheter. The blood circulatory system provides systemic spread of the pharmaceutical. Polyethylene glycol and other hydrophilic polymers have provided protection of the pharmaceuticals in the blood stream by preventing their interaction with blood components and increasing the circulatory time of the pharmaceuticals through preventing opsonization, phagocytosis and uptake by the reticuloendothelial system. For example, the enzyme adenosine deaminase has been covalently modified with polyethylene glycol to increase the circulatory time and persistence of this enzyme in the treatment of patients with adenosine deaminase deficiency.
The controlled release of pharmaceuticals after their administration is under intensive development. Pharmaceuticals have also been complexed with a variety of biologically-labile polymers to delay their release from depots. These polymers have included copolymers of poly(lactic/glycolic acid) (PLGA) (Jain, R. et al. Drug Dev. Ind. Pharm. 24, 703-727 (1998), ethylvinyl acetate/polyvinyl alcohol (Metrikin, DC and Anand, R, Curr Opin Ophthalmol 5, 21-29, 1994) as typical examples of biodegradable and non-degradable sustained release systems respectively.
Transdermal routes of administration have been effected by patches and ionotophoresis. Other epithelial routes include oral, nasal, respiratory, and vaginal routes of administration. These routes have attracted particular interest for the delivery of peptides, proteins, hormones, and cytokines which are typically administered by parenteral routes using needles. For example, the delivery of insulin via respiratory, oral, or nasal routes would be very attractive for patients with diabetes mellitus. For oral routes, the acidity of the stomach (pH less than 2) is avoided for pH-sensitive compounds by concealing peptidase-sensitive polypeptides inside pH-sensitive hydrogel matrix (copolymers of polyethyleneglycol and polyacrylic acid). After passing low pH compartments of gastrointestinal tract such structures swell at higher pH releasing thus a bioactive compound (Lowman AM et al. J. Pharm. Sci. 88, 933-937 (1999). Capsules have also been developed that release their contents within the small intestine based upon pH-dependent solubility of a polymer. Copolymers of polymethacrylic acid (Eudragit S, Rohm America) are known as polymers which are insoluble at lower pH but readily solubilized at higher pH, so they are used as enteric coatings (Z Hu et al. J. Drug Target., 7, 223, 1999).
Biologically active molecules may be assisted by a reversible formation of covalent bonds. Quite often, it is found that the drug administered to a patient is not the active form of the drug, but what is a called a prodrug that changes into the actual biologically active compound upon interactions with specific enzymes inside the body. In particular, anticancer drugs are quite toxic and are administered as prodrugs which do not become active until they come in contact with the cancerous cells (Sezaki, II., Takakura, Y., Hashida, M. Adv. Drug. Delivery Reviews 3, 193, 1989).
Liposomes were also used as drug delivery vehicles for low molecular weight drugs and macromolecules such as amphotericin B for systemic fungal infections and candidiasis. Inclusion of anti-cancer drugs such as adriamycin has been developed to increase their delivery to tumors and reducetheir access to other tissue sites (e.g. heart) thereby decreasing their toxicity. The use of pH-sensitive polymers in conjunction with liposomes represents another opportunity to modulate lipid bilayer permeability warranting thus triggered release of encapsulated drugs. For example, hydrophobically-modified N-isopropylacrylamide-methacrylic acid copolymer can render regular egg PC liposomes pH-sensitive by pH-dependent interaction of grafted aliphatic chains with lipid bilayer (O Meyer et al., FEBS Lett., 421, 61, 1998).
Gene And Nucleic Acid-Based Delivery
Gene or polynucleotide transfer is the cardinal process of gene therapy. The gene needs to be transferred across the cell membrane and enter the nucleus where the gene can be expressed. Gene transfer methods currently being explored include viral vectors and physical-chemical methods.
Viruses have evolved over millions of years to transfer their genes into mammalian cells. Viruses can be modified to carry a desired gene and become a “vector” for gene therapy. Using standard recombinant techniques, the harmful or superfluous viral genes can be removed and replaced with the desired normal genes. This was first accomplished with mouse retroviruses. The development of retroviral vectors were the catalyst that promoted current gene therapy efforts. However, they cannot infect all cell types very efficiently, especially in vivo. Other viral vectors based on Herpes virus are being developed to enable more efficient gene transfer into brain cells. Adenoviral and adenoassociated vectors are being developed to infect lung and other types of cells.
Besides using viral vectors, it is possible to directly transfer genes into mammalian cells. Usually, the desired gene is placed within bacterial plasmid DNA along with a mammalian promoter, enhancer, and other sequences that enable the gene to be expressed in mammalian cells. Several milligrams of the plasmid DNA containing all these sequences can be prepared and purified from the bacterial cultures. The plasmid DNA containing the desired gene can be incorporated into lipid vesicles (liposomes including cationic lipids such as Lipofectin) that then transfer the plasmid DNA into the target cell. Plasmid DNA can also be complexed with proteins that target the plasmid DNA to specific tissues, just as certain proteins are taken up (endocytosed) by specific cells. Also, plasmid DNA can be complexed with polymers such as polylysine and polyethylenimine. Another plasmid-based technique involves “shooting” the plasmid DNA on small gold beads into the cell using a “gun”. Finally, muscle cells in vivo have the unusual ability to take up and express plasmid DNA
Gene therapy approaches can be classified into direct and indirect methods. Some of these gene transfer methods are most effective when directly injected into a tissue space. Direct methods using many of the above gene transfer techniques are being used to target tumors, muscle, liver, lung, and brain. Other methods are most effective when applied to cells or tissues that have been removed from the body, genetically modified and then transplanted back into the body. Indirect approaches in conjunction with retroviral vectors are being developed to transfer genes into bone marrow cells, lymphocytes, hepatocytes, myoblasts and skin cells.
Gene Therapy And Nucleic Acid-Based Therapies
Gene therapy is a revolutionary advance in the treatment of disease. It is an approach for treating disease which is different from the conventional surgical and pharmaceutical therapies. Conceptually, gene therapy is a relatively simple approach. If someone has a defective gene, then gene therapy would fix the defective gene. The disease state would be modified by manipulating genes instead of the gene products. Although, the initial motivation for gene therapy was the treatment of genetic disorders, it is becoming increasingly apparent that gene therapy will be useful for the treatment of a broad range of acquired diseases such as cancer, infectious disorders (AIDS), heart disease, arthritis, and neurodegenerative disorders (Parkinsons and Alzheimers).
Gene therapy promises to take full-advantage of the major advances brought about by molecular biology. While biochemistry is mainly concerned with how the cell obtains the energy and matter that is required for normal function, molecular biology is mainly concerned with how the cell gets the information to perform its functions. Molecular biology wants to discover the flow of information in the cell. Using the metaphor of computers, the cell is the hardware while the genes are the software. In this sense, the purpose of gene therapy is to provide the cell with a new program (genetic information) so as to reprogram a dysfunctional cell to perform a normal function. The addition of a new cellular function is provided by the insertion of a foreign gene that expresses a foreign protein or a native protein at amounts that are not initially present in the patient.
The inhibition of a cellular function is provided by anti-sense approaches (that is acting against messenger RNA) and that includes using oligonucleotides complementary to the messenger RNA sequence and ribozymes. Messenger RNA (mRNA) is an intermediate in the expression of the DNA gene. The mRNA is translated into a protein. “Anti-sense” methods use a RNA sequence or an oligonucleotide that is made complementary to the target mRNA sequence and therefore binds specifically to the target messenger RNA. When this anti-sense sequence binds to the target mRNA, the mRNA is somehow destroyed or blocked from being translated. Ribozymes destroy a specific mRNA by a different mechanism. Ribozymes are RNA's that contain sequence complementary to the target messenger RNA plus a RNA sequence that acts as an enzyme to cleave the messenger RNA, thus destroying it and preventing it from being translated. When these anti-sense or ribozyme sequences are introduced into a cell, they would inactivate their specific target mRNA and reduce their disease-causing properties.
Several recessive genetic disorders are being considered for gene therapy. One of the first uses of gene therapy in humans has been used for the genetic deficiency of the adenosine deaminase (ADA) gene. Other clinical gene therapy trials have been conducted for cystic fibrosis, familial hypercholesteremia caused by a defective LDL-receptor gene and partial ornithine transcarbomylase deficiency. Both indirect and direct gene therapy approaches are being developed for Duchenne muscular dystrophy. Patients with this type of muscular dystrophy eventually die from loss of their respiratory muscles. Direct approaches include the intramuscular injection of naked plasmid DNA or adenoviral vectors.
A wide variety of gene therapy approaches for cancer are under investigation in animals and in human clinical trials. One approach is to express in lymphocytes and in the tumor cells cytokine genes that stimulate the immune system to destroy the cancer cells. The cytokine genes would be transferred into the lymphocytes by removing the lymphocytes from the body and infecting them with a retroviral vector carrying the cytokine gene. The tumor cells would be similarly genetically modified by this indirect approach to express cytokines within the tumor. Direct approaches involving the expression of cytokines in tumor cells in situ are also being considered. Other genes besides cytokines may be able to induce an immune response against the cancer. One approach that has entered clinical trials is the direct injection of HLA-B7 gene (which encodes a potent immunogen) within lipid vesicles into malignant melanomas in order to induce a more effective immune response against the cancer.
“Suicide” genes are genes that kill cells which express the gene. For example, the diphtheria toxin gene directly kill cells. The Herpes thymidine kinase (TK) gene kills cells in conjunction with acyclovir (a drug used to treat Herpes viral infections). Other gene therapy approaches take advantage of our knowledge of oncogenes and suppressor tumor genes- also known as anti-oncogenes. The loss of a functioning anti-oncogene plays a decisive role in childhood tumors such as retinoblastoma, osteosarcoma and Wilms tumor and may play an important role in more common tumors such as lung, colon and breast cancer. Introduction of the normal anti-oncogene back into these tumor cells may convert them back to normal cells. The activation of oncogenes also plays an important role in the development of cancers. Since these oncogenes operate in a “dominant” fashion, treatment will require inactivation of the abnormal oncogene. This can be done using either “anti-sense” or ribozyme methods that selectively inactivate a specific messenger RNA in a cell.
Gene therapy can be used as a type of vaccination to prevent infectious diseases and cancer. When a foreign gene is transferred into a cell and the protein is made, the foreign protein is presented to the immune system differently from simply injecting the foreign protein into the body. This different presentation is more likely to cause a cell-mediated immune response which is important for fighting latent viral infections such as human immunodeficiency virus (HIV causes AIDS), Herpes and cytomegalovirus. Expression of the viral gene within a cell simulates a viral infection and induces a more effective immune response by fooling the body that the cell is actually infected by the virus, without the danger of an actual viral infection.
One direct approach uses the direct intramuscular injection of naked plasmid DNA to express a viral gene in muscle cells. The “gun” has also been shown to be effective at inducing an immune response by expressing foreign genes in the skin. Other direct approaches involving the use of retroviral, vaccinia or adenoviral vectors are also being developed. An indirect approach has been developed to remove fibroblasts from the skin, infect them with a retroviral vector carrying a viral gene and transplant the cells back into the body. The envelope gene from the AIDS virus (HIV) is often used for these purposes. Many cancer cells express specific genes that normal cells do not. Therefore, these genes specifically expressed in cancer cells can be used for immunization against cancer.
Besides the above immunization approaches, several other gene therapies are being developed for treating infectious disease. Most of these new approaches are being developed for HIV infection and AIDS. Many of them will involve the delivery of anti-sense or ribozyme sequences directed against the particular viral messenger RNA. These anti-sense or ribozyme sequences will block the expression of specific viral genes and abort the viral infection without damaging the infected cell. Another approach somewhat similar to the ant-sense approaches is to overexpress the target sequences for these regulatory HIV sequences.
Gene therapy efforts would be directed at lowering the risk factors associated with atherosclerosis. Overexpression of the LDL receptor gene would lower blood cholesterol in patients not only with familial hypercholesteremia but with other causes of high cholesterol levels. The genes encoding the proteins for HDL (“the good cholesterol”) could be expressed also in various tissues. This would raise HDL levels and prevent atherosclerosis and heart attacks. Tissue plasminogen activator (tPA) protein is being given to patients immediately after their myocardial infarction to digest the blood clots and open up the blocked coronary blood vessels. The gene for tPA could be expressed in the endothelial cells lining the coronary blood vessels and thereby deliver the tPA locally without providing tPA throughout the body. Another approach for coronary vessel disease is to express a gene in the heart that produces a protein that causes new blood vessels to grow. This would increase collateral blood flow and prevent a myocardial infarction from occurring.
Neurodegenerative disorders such as Parkinson's and Alzheimer's diseases are good candidates for early attempts at gene therapy. Arthritis could also be treated by gene therapy. Several proteins and their genes (such as the IL-1 receptor antagonist protein) have recently been discovered to be anti-inflammatory. Expression of these genes in joint (synovial) fluid would decrease the joint inflammation and treat the arthritis.
In addition, methods are being developed to directly modify the sequence of target genes and chromosomal DNA. The delivery of a nucleic acid or other compound that modifies the genetic instruction (e.g., by homologous recombination) can correct a mutated gene or mutate a functioning gene.
Liver Gene Therapy
Liver is one of the most important target tissues for gene therapy given its central role in metabolism (e.g. lipoprotein metabolism in various hypercholesterolemias) and the secretion of circulating proteins (e.g. clotting factors in hemophilia). At least one hundred different genetic disorders could be corrected by liver-directed gene therapy. Their cumulative frequency is approximately one percent of all births. In addition, multifactorial disorders are also amenable to liver gene therapy. For example, diabetes mellitus could be treated by expressing the insulin gene within hepatocytes whose physiology may enable glucoseregulated insulin secretion. Acquired disorders such as chronic hepatitis (particularly important in Asia) could also be treated by polynucleotide-based liver therapies.
A variety of techniques have been developed to transfer genes into the liver. Jon Wolff and colleagues suggested the liver as a target tissue for gene therapy by demonstrating that primary hepatocytes could be efficiently infected with retroviral vectors (Wolff, et al., Proc. Natl. Acad. Sci. USA, 84:3444-3348. 1987). Cultured hepatocytes have been genetically modified by retroviral vectors and implanted back into livers of animals and humans (Grossman, et al., Nature Genet., 6:335-341. 1994, Chowdhury, et al., Science, 254:1802-1805. 1991, Ledley, et al., Somat. Cell Mol. Genet., 13:145-54. 1987). Retroviral vectors have also been delivered directly to livers in which hepatocyte division was induced by partial hepatectomy or cytokines (Bosch, et al., Journal of Clinical Investigation, 98:2683-7. 1996, Ferry, et al., Proc. Natl. Acad. Sci. USA, 88:8377-8381. 1991, Kaleko, et al., Hum. Gene Ther., 2:27-32. 1991, Kay, et al., Hum. Gene Ther., 3:641-7. 1992, Hafenrichter, et al., J. Surgical Res., 56:510-7. 1994, Rettinger, et al., Proc. Natl. Acad. Sci. USA, 91:1460-4. 1994). Injection of adenoviral vectors into the portal or systemic circulatory systems leads to high levels of foreign gene expression that is transient (Sullivan, et al., Human Gene Therapy, 8:1195-206. 1997, Jaffe, et al., Nature Genet., 1:372-378. 1992, Li, et al., Hum. Gene Ther., 4:403-409. 1993, Stratford-Perricaudet, et al., Hum. Gene Ther., 1:241-56. 1990). Long-term expression of AAV vectors or retroviral vectors derived from lentiviruses has been recently reported for liver and muscle (Snyder, et al., Nature Genetics, 16:270-6. 1997, Herzog, et al., Nature Medicine, 5:56-63. 1999, Linden and Woo, Nature Medicine, 5:21-22. 1999, Snyder, et al., Nature Medicine, 5:64-70, 1999) (Xiao, et al., Journal of Virology, 70:8098-8108, 1996, Fisher, et al., Nature Medicine, 3:306-312. 1997, Herzog, et al., Proceedings of the National Academy of Sciences of the United States of America, 94:5804-5809. 1997) (Kafri, et al., Nature Genetics, 17:214-317. 1997). Since AAV and retroviral vectors require administration directly in the liver or its blood vessels, hepatocyte-targeting peptides would improve their utility. Adenoviral vectors can target hepatocytes after peripheral vein injection but hepatocyte targeting would improve their safety and efficacy. Several groups are developing approaches to modify the targeting of retroviral and adenoviral vectors, that could readily incorporate the peptides discovered in this proposal (Reynolds and Curiel, The Development of Human Gene Therapy. Editor: T. Friedmann, Cold Spring arbor Press, pp. 111-130. 1999).
Non-viral transfer methods have included polylysine complexes of asialoglycoproteins that are systemically administered (Wu and Wu, Biochemistry, 27:887-92. 1988, Wu, et al., Journal of Biological Chemistry, 264:16985-7. 1989, Wilson, et al., Journal of Biological Chemistry, 267:963-7. 1992). Plasmid DNA expression in the liver has also been achieved via liposomes delivered by tail vein or intraportal routes (Kaneda, et al., J. Biol. Chem., 264:12126-12129. 1989, Kaneda, et al., Science, 243:375-378. 1989, Soriano, et al., Proc. Natl. Acad. Sci. USA, 80:7128-7131. 1993). One lab has shown that high levels of hepatocyte expression can be achieved by the injection of naked plasmid DNA (pDNA) into liver vessels or tail vein (Budker, et al., Gene Therapy, 3:593-8. 1996, Zhang, et al., Human Gene Therapy, 8:1763-72. 1997).
Paradigm For Development of Vectors
The current paradigm for the development of non-viral and viral vectors is to incorporate in a combinatorial fashion functional groups that enable particular transfer steps. These functional groups, initially discovered within proteins and viruses, serve as signals or “addresses” that interact with cellular components and cause the protein or virus to enter a particular sub-cellular compartment. These same signals can be incorporated into non-viral or viral vectors to enhance each transport step required for the therapeutic genes to eventually enter the cellular nucleus where the gene expresses its therapeutic function. These signals include surface molecules that resist inactivation in the blood, maintaining their ability to direct the vector toward target cells. After particle binding to the cell, the particle must contain other molecules to release the particle DNA into the cytoplasm. Finally, other functional groups can enable cytoplasmic transport to the nuclear membrane and traversal of the nuclear pore into the nucleus.
Polymers for Drug and Nucleic Acid Delivery
Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used in research for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells with an eventual goal of providing therapeutic processes. Such processes have been termed gene therapy or anti-sense therapy. One of the several methods of nucleic acid delivery to the cells is the use of DNA-polycation complexes. It has been shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents while small polycations like spermine are ineffective. The following are some important principles involving the mechanism by which polycations facilitate uptake of DNA.
Polycations provide attachment of DNA to the cell surface. The polymer forms a cross-bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells. As a result the main mechanism of DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis. Furthermore, polycations are a convenient linker for attaching specific receptors to DNA and as result, DNA- polycation complexes can be targeted to specific cell types.
Polycations protect DNA in complexes against nuclease degradation. This is important for both extra- and intracellular preservation of DNA. Gene expression is also enabled or increased by preventing endosome acidification with NH4Cl or chloroquine. Polyethylenimine, which facilitates gene expression without additional treatments, probably disrupts endosomal function itself. Disruption of endosomal function has also been accomplished by linking to the polycation endosomal-disruptive agents such as fusion peptides or adenoviruses.
Polycations can also facilitate DNA condensation. The volume which one DNA molecule occupies in a complex with polycations is drastically lower than the volume of a free DNA molecule. The size of a DNA/polymer complex is probably critical for gene delivery in vivo. In terms of intravenous injection, DNA needs to cross the endothelial barrier and reach the parenchymal cells of interest. The largest endothelia fenestrae (holes in the endothelial barrier) occur in the liver and have an average diameter of 100 nm. The trans-epithelial pores in other organs are much smaller, for example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20-30 nm. The size of the DNA complexes is also important for the cellular uptake process. After binding to the cells the DNA- polycation complex should be taken up by endocytosis. Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes and are of similar size in other cell types, DNA complexes smaller than 100 nm are preferred.
Condensation of DNA
A significant number of multivalent cations with widely different molecular structures have been shown to induce condensation of DNA.
Two approaches for compacting (used herein as an equivalent to the term condensing) DNA:
1. Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+,Fe3+, and natural or synthetic polymers such as histone H1, protamine, polylysine, and polyethylenimine. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized.
2. Polymers (neutral or anionic) which can increase repulsion between DNA and its surroundings have been shown to compact DNA. Most significantly, spontaneous DNA self-assembly and aggregation process have been shown to result from the confinement of large amounts of DNA, due to excluded volume effect.
Depending upon the concentration of DNA, condensation leads to three main types of structures:                1) In extremely dilute solution (about 1 ug/mL or below), long DNA molecules can undergo a monomolecular collapse and form structures described as toroid.        2) In very dilute solution (about 10 ug/mL) microaggregates form with short or long molecules and remain in suspension. Toroids, rods and small aggregates can be seen in such solution.        3) In dilute solution (about 1 mg/mL) large aggregates are formed that sediment readily.        
Toroids have been considered an attractive form for gene delivery because they have the smallest size. While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed. DNA molecules from 400 bp to genomic length produce toroids similar in size. Therefore one toroid can include from one to several DNA molecules. The kinetics of DNA collapse by polycations that resulted in toroids is very slow. For example DNA condensation by Co(NH3)6Cl3 needs 2 hours at room temperature.
The mechanism of DNA condensation is not clear. The electrostatic force between unperturbed helices arises primarily from a counterion fluctuation mechanism requiring multivalent cations and plays a major role in DNA condensation. The hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters. In a case of DNA-polymeric polycation interactions, DNA condensation is a more complicated process than the case of low molecular weight polycations. Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of 0.4. T4 DNA complexes with polyarginine or histone can form two types of structures; an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles. Both forms exist simultaneously in the same solution. The reason for the coexistence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules. The uneven distribution generates two thermodynamically favorable conformations.
The electrophoretic mobility of DNA-polycation complexes can change from negative to positive in excess of polycation. It is likely that large polycations don't completely align along DNA but form polymer loops that interact with other DNA molecules. The rapid aggregation and strong intermolecular forces between different DNA molecules may prevent the slow adjustment between helices needed to form tightly packed orderly particles.
Preparation of polycation-condensed DNA particles is of particular importance for gene therapy, more specifically, particle delivery such as the design of non-viral gene transfer vectors. Optimal transfection activity in vitro and in vivo can require an excess of polycation molecules. However, the presence of a large excess of polycations may be toxic to cells and tissues. Moreover, the non-specific binding of cationic particles to all cells forestalls cellular targeting. Positive charge also has an adverse influence on biodistribution of the complexes in vivo.
Several modifications of DNA-cation particles have been created to circumvent the nonspecific interactions of the DNA-cation particle and the toxicity of cationic particles. Examples of these modifications include attachment of steric stabilizers, e.g. polyethylene glycol, which inhibit nonspecific interactions between the cation and biological polyanions. Another example is recharging the DNA particle by the additions of polyanions which interact with the cationic particle thereby lowering its surface charge, i.e. recharging of the DNA particle. We have demonstrated that layering of polyelectrolytes can be achieved on the surface of DNA/polycation particles (V S Trubetskoy, A Loomis, J E Hagstrom, V G Budker, J A Wolff. Nucleic Acids Res. 27:3090-3095, 1999, incorporated herein by reference). Another example is cross-linking the polymers and thereby caging the complex (V S Trubetskoy, A Loomis, P M Slattum, J E Hagstrom, V G Budker, J A Wolff. Bioconjugate Chem. 10:624-628, 1999, incorporated herein by reference). Nucleic acid particles can be formed by the formation of chemical bonds and template polymerization. Trubetskoy et al used two types of polymerization reactions to achieve DNA condensation: step polymerization and chain polymerization (V S Trubetskoy, V G Budker, L I Hanson, P M Slattum, J A Wolff, L E Hagstrom. Nucleic Acids Res. 26:4178-4185, 1998) U.S. patent application Ser. No. 08/778,657 incorporated herein by reference.
The Use of pH-Sensitive Lipids, Amphipathic Compounds, and Liposomes for Drug and Nucleic Acid Delivery
After the landmark description of DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) [Felgner, P L, Gadek, T R, Holm, M, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA. 1987;84:7413-7417], a plethora of cationic lipids have been synthesized. Basically, all the cationic lipids are amphipathic compounds that contain a hydrophobic domain, a spacer, and positively-charged amine. The hydrophilic domains are typically hydrocarbon chains such as fatty acids derived from oleic or myristic acid. The hydrocarbon chains are often joined either by ether or ester bonds to a spacer such as glycerol. Quaternary amines often compose the cationic groups. Usually, the cationic lipids are mixed with a fusogenic lipid such as DOPE (dioleoyl phosphatidyl ethanolamine) to form liposomes. The mixtures are mixed in chloroform that is then dried. Water is added to the dried lipid film and unilamellar liposomes form during sonication. Multilamellar cationic liposomes and cationic liposomes/DNA complexes prepared by the reverse-phase evaporation method have also been used for transfection. Cationic liposomes have also been prepared by an ethanol injection technique.
Several cationic lipids contain a spermine group for binding to DNA. DOSPA, the cationic lipid within the LipofectAMINE formulation (Life Technologies) contains a spermine linked via a amide bond and ethyl group to a trimethyl, quaternary amine [Hawley-Nelson, P, Ciccarone, V and Jessee, J. Lipofectamine reagent: A new, higher efficiency polycationic liposome transfection reagent. Focus 1993; 15:73-79]. A French group has synthesized a series of cationic lipids such as DOGS (dioctadecylglycinespermine) that contain spermine [Remy, J-S, Sirlin, C, Vierling, P, et al. Gene transfer with a series of lipophilic DNA-binding molecules. Bioconjugate Chem. 1994;5:647-654]. DNA has also been transfected by lipophilic polylysines which contain dipalmotoylsuccinylglycerol chemically-bonded to low molecular weight (˜3000 MW) polylysine [Zhou, X Kilbanov, A and Huang, L. Lipophilic polylysines mediate efficient DNA transfection in mammalian cells. Biochim. Biophys. Acta 1991;1065:8-14. Zhou, X and Huang, L. DNA transfection mediated by cationic liposomes containing lipopolylysine: Characterization and mechanism of action Biochim. Biophys. Acta 1994;1195-203].
Other studies have used adjuvants with the cationic liposomes. Transfection efficiency into Cos cells was increased when amphiphilic peptides derived from influenza virus hemagglutinin were added to DOTMA/DOPE liposomes [Kamata, H, Yagisawa, H, Takahashi S, et al. Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection. Nucleic Acids Res. 1994;22:536-537]. Cationic lipids have been combined with galactose ligands for targeting to the hepatocyte asialoglycoprotein receptor [Remy, J-S, Kichier, A, Mordvinov, V, et al. Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: A stage toward artificial viruses. Proc. Nail. Acad. Sci. USA 1995;92:1744-1748]. Thiol-reactive phospholipids have also been incorporated into cationic lipid/pDNA complexes to enable cellular binding even when the net charge of the complex is not positive [Kichler, A, Remy, J-S, Boussif, O, et al. Efficient gene delivery with neutral complexes of lipospemmine and thiol-reactive phospholipids. Biochem. Biophys. Res. Comm. 1995;209:444-450]. DNA-dependent template process converted thiol-containing detergent possessing high critical micelle concentration into dimeric lipid-like molecule with apparently low water solubility (J P Behr and colleagues).
Cationic liposomes may deliver DNA either directly across the plasma membrane or via the endosome compartment. Regardless of its exact entry point, much of the DNA within cationic liposomes does accumulate in the endosome compartment. Several approaches have been investigated to prevent loss of the foreign DNA in the endosomal compartment by protecting it from hydrolytic digestion within the endosomes or enabling its escape from endosomes into the cytoplasm. They include the use of acidotropic (lysomotrophic), weak amines such as chloroquine that presumably prevent DNA degradation by inhibiting endosomal acidification [Legendre, J. & Szoka, F. Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: Comparison with cationic liposomes. Pharmaceut. Res. 9, 1235-1242 (1992)]. Viral fusion peptides or whole virus have been included to disrupt endosomes or promote fusion of liposomes with endosomes, and facilitate release of DNA into the cytoplasm [Kamata, H., Yagisawa, H., Takahashi, S. & Hirata, H. Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection. Nucleic Acids Res. 22, 536-537 (1994). Wagner, E., Curiel, D. & Cotten, M. Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis. Advance Drug Delivery Reviews 14, 113-135 (1994)].
Knowledge of lipid phases and membrane fusion has been used to design potentially more versatile liposomes that exploit the endosomal acidification to promote fusion with endosomal membranes. Such an approach is best exemplified by anionic, pH-sensitive liposomes that have been designed to destabilize or fuse with the endosome membrane at acidic pH [Duzgunes, N., Straubinger, R. M., Baldwin, P. A & Papahadjopoulos, D. PH-sensitive liposomes. (eds Wilschub, J. & Hoekstra, D.) p. 713-730 (Marcel Dekrer INC, 1991)]. All of the anionic, pH-sensitive liposomes have utilized phosphatidylethanolamine (PE) bilayers that are stabilized at non-acidic pH by the addition of lipids which contain a carboxylic acid group. Liposomes containing only PE are prone to the inverted hexagonal phase (HII). In pH-sensitive, anionic liposomes, the carboxylic acid's negative charge increases the size of the lipid head group at pH greater than the carboxylic acid's pK and thereby stabilizes the phosphatidylethanolamine bilayer. At acidic pH within endosomes, the uncharged or reduced charge species is unable to stabilize the phosphatidylethanolamine-rich bilayer. Anionic, pH-sensitive liposomes have delivered a variety of membrane-impermeant compounds including DNA. However, the negative charge of these pH-sensitive liposomes prevents them from efficiently taking up DNA and interacting with cells; thus decreasing their utility for transfection. We have described the use of cationic, pH-sensitive liposomes to mediate the efficient transfer of DNA into a variety of cells in culture.
The Use of pH-Sensitive Polymers for Drug and Nucleic Acid Delivery
pH-sensitive polymers have found broad application in the area of drug delivery exploiting various physiological and intracellular pH gradients for the purpose of controlled release of drugs (both low molecular weight and polymeric). pH sensitivity can be broadly defined as any change in polymer's physico-chemical properties over certain range of pH. More narrow definition demands significant changes in polymer's ability to retain (release) bioactive substance (drug) in physiologically tolerated pH range (usually pH 5.5-8). pH-sensitivity presumes the presence of ionizable groups in the polymer (polyion). All polyions can be divided into three categories based on their ability to donate or accept protons in aqueous solutions: polyacids, polybases and polyampholytes. Use of pH-sensitive polyacids in drug delivery applications usually relies on their ability to become soluble with the pH increase(acid/salt conversion), to form complex with other polymers over change of pH or undergo significant change in hydrophobicity/hydrophilicity balance. Combinations of all three above factors are also possible.
Copolymers of polymethacrylic acid (Eudragit S, Rohm America) are known as polymers which are insoluble at lower pH but readily solubilized at higher pH, so they are used as enteric coatings designed to dissolve at higher intestinal pH (Z Hu et al. J. Drug Target., 7, 223, 1999). Typical example of pH-dependent complexation is copolymers of polyacrylate(graft)ethyleneglycol which can be formulated into various pH-sensitive hydrogels which exhibit pH-dependent swelling and drug release (F Madsen et al., Biomaterials, 20, 1701, 1999). Hydrophobically-modified N-isopropylacrylamide-methacrylic acid copolymer can render regular egg PC liposomes pH-sensitive by pH-dependent interaction of grafted aliphatic chains with lipid bilayer (O Meyer et al., FEBS Lett., 421, 61, 1998). Polymers with pH-mediated hydrophobicity (like polyethylacrylic acid) can be used as endosomal disruptors for cytoplasmic drug delivery (N Murthy et al. J. Controlled Release 61, 137, 1999).
Polybases have found broad applications as agents for nucleic acid delivery in transfection/gene therapy applications due to the fact they are readily interact with polyacids. Typical example is polyethyleneimine (PEI). This polymer secures nucleic acid electrostatic adsorption on the cell surface followed by endocytosis of the whole complex. Cytoplasmic release of the nucleic acid occures presumably via so called “proton sponge” effect according to which pH-sensitivity of PEI is responsible for endosome rupture due to osmotic swelling during its acidification (O Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297, 1995). Cationic acrylates possess the similar activity (for example, poly-((2-dimethylamino)ethyl methacrylate) (P van de Wetering et al. J. Controlled Release 64, 193, 2000). However, polybases due to their polycationic nature pH-sensitive polybases have not find broad in vivo application so far. They exhibit acute systemic toxicity in vivo presumably mostly of colloid nature (JH Senior, Biochim. Biophys. Acta, 1070, 173, 1991). Milder polybases (for example, linear PEI) are better tolerated and can be used systemically for in vivo gene transfer (D Goula et al. Gene Therapy 5, 712, 1998).
Blood Interactions of Delivery Complexes
Blood interactions are of importance for in vivo delivery of drugs and genes. Retroviral vectors are inhibited in human blood through complement (C) activation(Russell, et al., Human Gene Therapy, 6:635-641. 1995, Welsh, et al., Nature, 257:612-614. 1975, Rother, et al., Hum. Gene Ther., 6:429-435. 1995). Retroviral vectors produced in non-human cell lines contain galactosyl(α1-3)galactosyl(αGal) terminal sugars within glycolipids and glycoproteins(Cosset, et al., J. Virol., 69:7430-7436. 1995). Humans lack this sugar structure and produce antibodies against it (sensitized presumably by gut flora). Retroviral vectors produced in αGal-negative cell lines do not suffer this problem. Similar findings have been made for VSV, HIV-2, human foamy viruses and the vectors derived from them (Takeuchi, et al., J. Virol., 71:6174-6178. 1997). Another mechanism for retroviral inactivation involves C activation via direct binding of C1q to the p15 (envelope) transmembrane protein(Welsh, et al., Nature, 257:612-614. 1975, Pensiero, et al., Human Gene Therapy, 7:1095-1101. 1996, Bartholomew and Esser, Biochemistry, 19:2847-2853. 1980). Another viral vector system, baculovirus vectors (which can transduce mammalian hepatocytes), is inhibited by serum but not by C inactive serum (heat treated or depleted in C3 or C4)(Sandig, et al., Human Gene Therapy, 7:1937-1945. 1996). Other factors such as chondroitin sulfates within pleural effusions inhibit retroviral vector gene transfer(Batra, et al., J. Biol. Chem., 272:11736-11741. 1997).
Of relevance to the development of non-viral vectors, is the vast literature on the interactions of the C system and other serum factors with liposomes (Szebeni, Crit. Rev Therap. Drug Carrier Systems, 15:57-88. 1998). Opsonization of liposomes by serum proteins plays an important role in their clearance by the reticuloendothelial system (RES). For example, clearance of negatively-charged liposomes is aided by β2-glycoprotein I binding (Chonn, et al., J. Biol. Chem., 270:25845-25849. 1995). Generally, neutral liposomes are poor C activators but they are cleared similarly to anionic liposomes in vivo, possibly because they absorb anionic serum proteins in vivo (Devine and Bradley, Advanced Drug Delivery Reviews, 32:19-29. 1998). “Plain” liposomes (phospholipid/cholesterol bilayers without antigenic components) bind several serum proteins including albumin, IgG, extracellular matrix proteins (fibronectin, laminin, serum amyloid protein), clotting factors, apolipoproteins, β2-glycoprotein-1, C reactive protein (CRP), α2-macroglobulin, and C factor such as C1q and C3 (Senior, Crit. Rev. Ther. Drug Carrier Syst., 3:123. 1987, Scherphof, et al., Liposome Technology, 205. 1984, Juliano, Liposomes, 53. 1983). Apo E binds to both anionic and neutral liposomes, but only the small, neutral liposomes had reduced liver targeting in apo E-deficient mice (Scherphof and Kamps, Advanced Drug Delivery Reviews, 32:81-97. 1998). This suggests that apo E-binding is important for the liver targeting of neutral but not anionic liposomes. Anionic liposomes activate C via the classical pathway starting with C1q (without anti-phosholipid antibodies), which leads to deposition of C3b and iC3b on the liposome's surface (Liu, et al., Biochim. Biophys. Acta, 1235:140-146. 1995) (Devine and Bradley, Advanced Drug Delivery Reviews, 32:19-29. 1998). Cationic liposomes activate C via the alternative pathway in human serum but weakly in rat serum. Another pathway is due to CRP binding of phosphocholine and galactosyl residues within liposomes (Volanakis and Narkates, J. Immunology, 126:1820-1825. 1981). The clearance of liposomes is affected by several other factors such as their size, fluidity, packing, and cholesterol content. In summary, the clearance of conventional liposomes is delayed by using small, neutral, unilamellar liposomes containing rigid bilayers (disteraroyl phosphatidylcholine or sphingomylein and cholesterol) (Lasic and Martin, Pharmacology and Toxicology: Basic and Clinical Aspects, 1995). Besides affecting delivery, C activation can also release anaphylatoxins (e.g., C3a, C4a, and C5a) that activate mast cells, basophils and platelets causing respiratory, blood pressure and dermatologic sequelae.
Many of this field's concepts and experimental methods are now being extended to the use of cationic lipids for DNA transfer. Serum inhibits the transfection ith the use of several types of cationic lipids by modifying the DNA/cationic lipid complexes (Escriou, et al., Biochim. Biophys. Acta, 1368:276-288. 1998, Zelphati and Szoka, Pharmaceutical Research, 13:1367-1372. 1996, Senior, et al., Biochimica et Biophysica Acta, 1070:173-9. 1991, Felgner, et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417. 1987, Zelphati, et al., Biochim. Biophys. Acta, 1390:119-133. 1998, Li, et al., Gene Therapy, 5:930-937. 1998) (Yang and Huang, Gene Therapy, 5:380-387. 1998, Yang and Huang, Gene Therapy, 4:950-960. 1997). Cationic lipid/DNA complexes activate C but it does not affect in vivo gene transfer (Barron, et al., Human Gene Therapy, 9:315-323. 1998). DNA/cationic lipid complexes made with GL-67 did not activate C because the complexes contain neutral lipids and have charge close to neutrality (Scheule, et al., Human Gene Therapy, 8:689-707. 1997, Plank, et al., Human Gene Therapy, 7:1437-1446. 1996).
Prevention of Unfavorable Interactions
On the basis of the above paradigm, we could borrow from the arsenal of cellular and viral approaches for preventing blood inactivation. For C mediated pathways, a variety of membrane (DAF, MIR1, CR1, MCP) and fluid-phase (C1 inh, C4bp, factor I, factor H) proteins prevent C activation on native cells(Devine and Bradley, Advanced Drug Delivery Reviews, 32:19-29. 1998). Viruses borrow these C inhibitory factors from cells as a “cloak” to prevent C activation. For example, HIV type I uses decay-accelerating factor (CD55) to inhibit C activation(Marschang, et al., Eur. J. Immunol., 25:285-290. 1995). Vectors could incorporate new chimeric or modified C regulatory proteins that are being developed to inhibit C activation (e.g., solubilized C3 convertase inhibitor, modified CD55)(Ryan, Nature Medicine, 1:967-968. 1995).
A “Dysopsonin” hypothesis has been proposed in which serum proteins can bind to foreign particles and prolong their blood circulation(Moghimi, et al., Biochim. BIophys. Acta, 1179:157-165. 1993). For example, Moghimi and colleagues have reported that two serum proteins prevented the uptake of poloxamine (a block co-polymer of polyoxyethylene and polyoxypropylene)-coated microspheres by isolated liver sinusoidal cells but did not identify the proteins. Our preliminary studies indicate that C-reactive protein (CRP) binding to T7 phage can prolong their blood circulation by preventing phage inactivation by C. This work also suggests a new approach for targeting in which natural serum proteins selectively adhere to the delivery particle and provide it with targeting properties.
In terms of artificial delivery systems, incorporation of specific glycoplipids such as GM1 ganglioside, cerebroside sulfate, or phosphatidylinositol or PEG (polyethylene glycol) prolongs the circulation time of liposomes in the blood by providing “steric stabilization”(Lasic and Martin, Pharmacology and Toxicology: Basic and Clinical Aspects, 1995). PEG and other hydrophilic polymers have been incorporated into a variety of polycation- and cationic lipid-containing gene transfer systems (Eastman, et al., Human Gene Therapy, 8:765-73. 1997, Toncheva, et al., Biochim. Biophys. Acta, 1380:354-368. 1998, Wolfert, et al., Human Gene Therapy, 7:2123-33. 1996, Astafieva, et al., FEBS Lett., 389:278-80. 1996, Meyer, et al., J. Biol. Chem., 273:15621-7. 1998, Katayose and Kataoka, J. Pharm. Sci., 87:160-163. 1998, Maruyama, et al., Bioconj. Chem., 8:735-742. 1997, Ferdous, et al., J. Pharm. Sci., 87:1400-1404. 1998, Ferdous, et al., Nucleic Acid Res., 26:3949-3954. 1998, Asayama, et al., Bioconj. Chem., 9:476-481. 1998, Vinogradov, et al., Bioconj. Chem., 9:805-812. 1998, Choi et al., Bioconjugate Chem., 9:708-718. 1998). Despite the promise of PEG, its use can be challenging. For example, it's attachment can interfere with cell surface receptor interactions and endocytosis (Lasic and Martin, Pharmacology and Toxicology: Basic and Clinical Aspects, 1995, Lasic, 1997). PEGylation of adenoviral vectors also interferes with transduction. Sialic acid-containing molecules such as glycophorin (a major RBC sialoglycoprotein), GM3 (a principal sialoglycolipid), GM1 and heparin have been incorporated into liposomes in order to mimic the membrane molecules on cells that inhibit C activation(Okada, et al., Immunology, 48:129. 1983, Okada, et al., J. Immunol., 134:3316. 1985, Michalek, et al., J. Immunol., 140:1581. 1988, Michalek, et al., J. Immunol., 140:1588. 1988, Shichijo and Alving, Biochim. Biophys. Acta., 858:118. 1986). The inhibitory effect of these compounds was abrogated by removal of sialic acid with neuramimidase digestion. Sialic acid surfaces preferentially bind factor H which inhibits the alternative pathway by preventing factor B binding to C3b.
Phage Display Systems, A Powerful Approach for Enhancing Gene Delivery:
The idea of using peptide ligands for targeting drug and gene delivery vehicles (Cotter, M. & Wagner, E. Receptor-mediated gene delivery strategies. (eds Friedmann, T.) p. 261-279 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1999)., Reynolds, P. N. & Curiel, D. T. Strategies to adapt adenoviral vectors or gene therapy applications: Targeting and integration. (eds Friedmann, T.) p. 111-130 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1999)) and for constructing biocompatible materials (Healy, et al., Ann. N. Y. Acad. Sci., 875:24-35. 1999, Shakesheff, et al., Journal of Biomaterials Science, Polymer Edition, 9:507-18. 1998) is widely accepted due to its conceptual and technical simplicity. A number of peptides with tissue targeting properties were selected both in vitro and in vivo by using the peptide libraries displayed at the amino-terminus of the filamentous phage coat proteins pIII or pVIII (Healy, et al., Ann. N.Y. Acad. Sci., 875:24-35. 1999, Shakesheff, et al., Journal of Biomaterials Science, Polymer Edition, 9:507-18. 1998, Pasqualini and Ruoslahti, Nature, 380:364-6. 1996, Pasqualini et al., Nat. Biotechnol., 15:542-546. 1997, Rajotte, et al., J. Clin. Invest., 102:430-437. 1998, Rajotte and Ruoslahti, J. Biol. Chem., 274:11593-8. 1999, Samoylova and Smith, Muscle Nerve, 22:460-6. 1999, Pasqualini, Quart. J. Nucl. Med., 43:159-62. 1999, Koivunen et al., Meth. Mot. Biol. 129:3-17. 1999, Koivunen et al., Nature Biotech., 17:768-74. 1999, Kassner et al., Biochem. Biophys. Res. Comm., 264:921-8. 1999, Koivunen et al., J. Nuct Med., 40:883-8. 1999, Ivanenkov et al., Biochim. Biophys. Acta, 1448:463-72. 1999, Ivanenkov et al., Biochim. Biophys. Acta, 1448:450-62. 1999, Barinaga, Science, 279:3234. 1998, Arap et al., 279:377-80. 1998, Folkman, Nature Biotech. 15:510).
Libraries of small peptides have been used to map epitopes, protein—protein interactions, protease inhibitors, integrin ligands, and receptor agonists and antagonists (Cwirla, et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382. 1990, Scott and Smith, Science, 249:386-390. 1990, O'Neil et al., Proteins, 14:509-15. 1992, Smith, et al., J. Biol. Chem., 270:6440-9. 1995, Doorbar and Winter, J. Mol. Biol., 244:361-9. 1994, Hong and Boulanger, EMBO J., 14:4714-27. 1995, Ferrer and Harisson, J. Virology, 73:5795-802. 1999, Kola et al., Mol. Immunol., 36:145-52. 1999). Phages displaying larger polypeptides such as antibodies, hormones, enzymes, and DNA-binding proteins have also been screened (Lowman, et al., Biochem., 30:10832-8. 1991, Rebar and Pabo, Science, 263:671-3. 1994, Soumillion, et al., J. Molec. Biol., 237:415-22. 1994, Roberts, et al., Proc. Natl. Acad. Sci. USA, 89:2429-33. 1992).
Of note is the work that is using phage display libraries to develop gene transfer methods (Russell, Nature Med., 2:276-277. 1996). Peptides that bind to shared receptors on different cell lines have been obtained by alternating rounds of biopanning among the different cells (Goodson, et al., Proc. Natl. Acad. Sci. USA, 91:7129-33. 1994). Following injection of a peptide library into the tail vein of mice, brain and kidney-specific peptide sequences were identified and used for specific in vivo targeting of red blood cells (Pasqualini and Ruoslahti, Nature, 380:364-366. 1996). A phage containing the integrin-binding RGD peptide was internalized by cultured human laryngeal epithelial cells (Hart, et al., J. Biol. Chem., 269:12468-12474. 1994). Using bacteriophage, a peptide that binds α9β1-integrin was identified for incorporation into non-viral vectors that are able to transfect airway epithelia (Schneider, et al., FASEB Letters, 429:269-273. 1998). Another group screened an M13 (pIII, 20-mer) library and selected for phage that can be internalized by cells in culture and that had affinity for muscle cells (Barry, et al., Nature Med., 2:299-305. 1996). Recently, bacteriophage libraries were used to identify peptides that can target GST-fusion proteins to lung endothelium (Rajotte, et al., J. Clin. Invest., 102:430-437. 1998). Interestingly, a pIII/M13 27-mer peptide display phage library was used to identify a C3-binding peptide (by panning for C3b-binding phages) that inhibits human C but not rat or mouse C (Sahu, et al., J. Immunol., 157:884-891. 1996).
Of relevance is a study that selected intraperitoneally injected λ bacteriophage for long circulation in the blood(Merril, et al., Proc. Natl. Acad. Sci. USA, 93:3188-3192. 1996). Two bacteriophage clones were selected. One had a glutamic acid to lysine substitution in position 158 (not the carboxy terminus) of the major λ capsid head protein E. Another clone had this same mutation plus an uncharacterized mutation in the λ capsid head protein D. The mechanism by which these mutations enabled prolonged circulation was not characterized.
Methods of Producing Antibodies
The techniques currently used in production of antibodies fall into several groups. The oldest approach uses serum from immunized animals as a source of antibodies The antigen can be injected in different forms, at different locations and at different times into animals of different species. The resultant serum can be used as is or antibodies with a different degree of purity can be isolated from serum using various precipitation, extraction, chromatographic and electrophoretic techniques or their combinations (Harlow and Lane. Antibodies:a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988).
Antibodies can be also produced in vitro, using the hybridoma technology by isolating B-cells from the animals pre-immunized with a particular antigen and growing them in culture. The isolated cells are immortalized by fusing them with myeloma cells that do not produce immunoglobulins of their own. The resultant hybrids are cloned and the clones that secret antibodies against the antigen of interest are selected and propagated further either in culture or as ascites. The secreted antibodies are monoclonal antibodies (Yokoyama WM. Production of monoclonal antibodies. (Eds. Coligan et al.,) Current Protocols in Immunology. New York: John Willey and Sons, 1995:2.5.1-2.5.17, Harlow and Lane. Antibodies:a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 1988,).
An alternative apprach to hybridoma technology is based on the use of gene libraries and expression systems. This allows to avoid labor-intensive immunizations of animals and screening of supernatants. Besides, this approach allows to circumvent tolerance. VH and VL libraries are prepared separately and then combined into a combinatorial library by cleaving, mixing and religating the libraries at a restriction site (Huse et al., Science, 246:1275-1281. 1989, Clackson et al., Nature, 352:624-28. 1991). VH and VL can both be expressed on one covalent polypeptide (Clackson et al., Nature, 352:624-28. 1991).
The power of the combinatorial approach is considerably enhanced by using phage display libraries, where VH and VL genes are expressed on the surface of phage particles as fusion protein with the phage coat protein. This approach permits screening of a large number of sequences (Clackson et al., Nature, 352:624-28. 1991, McCafferty et al., Nature, 348:552-554. 1990). The selection can be started with as many as 1010 clones prepared from “naive” B-cells. Selected combinations of VH and VL genes can be recombined into “hierachic libraries” and selection repeated (McCafferty et al., Nature, 348:552-554. 1990).
The increase in the affinity can also be achieved by mutating selected clones using such techniques as growing phage in the mulD5 E. coli strain with an error-prone DNA-polymerase III, “shuffling” of selected sequences, error-prone PCR and site-directed mutagenesis (Low et al., J. Mol. Biol., 260:359-368. 1996, Thompson et al., J. Mol. Biol., 256:77-88 1996).
Another important development in production of monoclonal antibodies was designing antibodies with some or all structure derived from human immunoglobulins. Such antibodies have lower immunogenicity in humans and, therefore, higher therapeutic potential. Several approaches have been taken based of fusion of human cells with animal myelomas or with human tumor cells or immortalization of human cells by using Epstein-Barr virus (Cole et al., Proc. Natl. Acad. Sci., 80:2026-2030. 1983, Olsson and Kaplan, Methods Enzymol., 92:3-16. 1983, Seigneurin et al., Science, 221:173-175). The recombination of selected animal variable regions with human constant regions gives so-called chimeric antibodies (Morrison SL. Science, 229:1202-1207. 1985, Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855. 1984). The additional substitution of animal framework sequences in the variable regions by human ones gives so called “humanized” antibodies (Jones et al., Nature, 321:522-525. 1986). Some additional engineering work is typically required to optimize the exposure of selected portions of the animal variable region in the new scaffold. Completely human antibodies can be produced now in transgenic mice (Lonberg et al., Nature, 368:856-859. 1994, Green et al., Nat. Genet., 7:13-21. 1994).
Single-stranded antibodies can be produced in a soluble form in E. coli. The incorporation of specific, “dimerizing” sequences into single-stranded antibodies with different specificities allows one to produce bispecific antibodies composed of two connected antibody molecules. One of the most powerful uses of such molecules is redirecting cytolytic cells to defined targets (Karpovsky et al., J. Exp. Med., 160:1686-1701. 1984, Titus et al., J. Immunol., 138:4018-4022. 1987). Another interesting application of bi-specific antibodies is changing the viral tropism (Wickham et al., J. Virol., 70:6931-6838).
Uses of Antibodies for Therapeutic Purposes A number of in vivo applications for monoclonal antibodies have been developed (Waldmann TA, Science, 252:1657-62. 1991, Berkower I., Curr. Opin. Biothechnol., 7:622-8. 1988). Leukemias and lymphomas have so far been the favorite targets for in vivo therapy based on the use of monoclonal antibodies (Appelbaum FR., Sem. Hemat., 36(4 Suppl. 6):2-8. 1999, Bendandi and Longo, Curr. Opin. Oncol., 11:343-50. 1999). Numerous attempts to treat other types of cancer have been undertaken as well (Reviewed by Wawrzyczak E J, Antibody therapy, Oxford, UK:Bios Scientific Publishers. 1995, Weiner L M., Sem Oncol., 26 (4 Suppl. 14):43-51. 1999, Sem. Oncol. (4 Suppl. 12):41-50). The antibodies can be used either by themselves, relying on the antibody effector functions, or as conjugates with various toxins and radionuclides (Weiner LM., Sem. Oncol., 26 (4 Suppl. 14):43-51. 1999, Sem. Oncol. (4 Suppl. 12):41-50). Antibodies have been also used for a variety of other applications, such as targeting platelets (Gensini et al., Am. Heart J. (2 Pt. 2), 138:171-6. 1999), blocking T-lymphocytes in rejection reactions (Ortho Multicenter Transplant Study Group, N. Engl. J. Med., 313:337-342), intercepting LPS for treatment of sepsis, blocking IL-6 receptor for treatment of multiple myeloma, and membrane IgE for treatment of allergy (Reviewed by Berkower I., Curr. Opin. Biothechnol., 7:622-8. 1988).
Uses of Antibodies for Diagnostic Purposes Most current applications of antibodies serve diagnostic rather than curative purposes. In vitro, they are widely used in RIA and ELISA measurements of substances in biological fluids, from hormones to toxins. They are also indispensable in flow cytometric assays. In vitro, the antigens to be identified are typically fractionated prior to the reaction with antibodies and immobilized on special supports. The antigen-antibody complexes are visualized by using secondary antibodies conjugated with fluorescent or electron-scattering labels or enzymes that digest specific substrate and cause the location precipitation of resultant products. In vivo, antibodies are used as tumor-imaging reagents (Collier et al., Radiology, 185:179-186, 1992).